In the context of gene therapy in a mammal, it is important to monitor the localization of a transgene. Where the transgene encodes a therapeutic polypeptide, such as a protein targeted to kill cancer cells, it is advantageous to have information as to the location, that is, the specific organs, tissues and/or cells which are expressing the polypeptide. There is a need in the art for methods and materials that permit the monitoring of tissue- or cell-specific transgene expression without the requirement to sample and directly test genetically modified cells or tissues.
The invention contemplates a method of monitoring the location of a transgene in a mammal, comprising the steps of (a) administering to a mammal in need thereof nucleic acid comprising a transgene and a sequence encoding a sodium-iodide symporter (NIS), wherein expression of NIS in cells permits cellular uptake of iodine (b) administering to a mammal labeled iodine in an amount sufficient to permit transport of the labeled iodine by NIS and detection of transported labeled iodine; and (c) detecting the location of the transported labeled iodine in the mammal as an indication of the location of the transgene.
In some embodiments, the step of detecting is performed quantitatively to determine the amount of transported labeled iodine in a mammal. The location of the transported labeled iodine is indicative of the location of NIS, whereby the location of NIS is indicative of the location of the transgene.
The invention also provides a method of monitoring the location of a transgene in a mammal, comprising the steps of (a) transfecting a host cell ex vivo with nucleic acid comprising a transgene and a sequence encoding and expressing NIS, wherein the NIS permits cellular uptake of iodine by the host cells; (b) introducing the transfected host cell into a mammal; (c) administering to the mammal labeled iodine in an amount sufficient to permit transport of the labeled iodine by NIS and detection of transported labeled iodine; and (d) determining the location of transported labeled iodine in the mammal; whereby the location of transported labeled iodine is indicative of the location of the transgene.
In preferred embodiments, the labeled iodine is radioactive iodine.
The invention also provides a nucleic acid construct comprising a chimeric gene comprising the transgene and the sequence encoding an NIS, wherein the chimeric gene also comprises a sequence encoding a protease-cleavable linker between the transgene and the sequence encoding NIS.
In a further embodiment, the sequence encoding the protease-cleavable amino acid linker comprises a sequence encoding an auto-cleaving sequence.
The invention also provides a nucleic acid construct comprising a first promoter operably associated with the transgene and a second promoter operably associated with the sequence encoding NIS.
The invention further provides a nucleic acid construct comprising a chimeric gene comprising a transgene and the sequence encoding NIS, wherein the chimeric gene also comprises between the transgene and the sequence encoding NIS, a sequence encoding an internal ribosome entry site.
In a preferred embodiment, the sequence encoding a protease cleabvable linker is attached to the 5xe2x80x2 end of the transgene.
In another preferred embodiment, the sequence encoding the protease-cleavable linker is attached to the 3xe2x80x2 end of the transgene.
In a preferred series of embodiments, the protease cleavable linker is cleaved by furin, or is identical to a linker present in a cytoplasmic protein.
In another series of preferred embodiments, the transgene encodes a fusogenic polypeptide, the fusogenic polypeptide encodes a viral fusion protein, the fusogenic polypeptide encodes a measles virus H glycoprotein, or the fusogenic polypeptide encodes a gibbon ape leukemia virus envelope glycoprotein.
The invention additionally provides a host cell comprising (a) a nucleic acid construct comprising a sequence encoding a transgene and a sequence encoding a sodium-iodide symporter (NIS), wherein the chimeric gene also comprises a sequence encoding a protease-cleavable linker between the transgene and the sequence encoding NIS; (b) a construct comprising a first promoter operable associated with the transgene and a second promoter is operable associated with the sequence encoding NIS; or (c) a construct comprising a chimeric gene comprising the transgene and the sequence encoding NIS, wherein the chimeric gene also comprises between the transgene and the sequence encoding NIS, a sequence encoding an internal ribosome entry site.
The invention further provides a kit comprising, in a ready to use format, one or more of the nucleic acid constructs described above, and one or more reagents for monitoring the location of the transported labeled iodine.
The invention still further provides a kit comprising, in a ready to use format, a host cell transfected; with one or more of the nucleic acid constructs described above, and one or more reagents for monitoring the location of the transported labeled iodine.
In a preferred embodiment, the reagents of the kit include labeled iodine.
In a preferred embodiment, the reagents of the kit include radioactive iodine.
The invention thus provides the art with methods and materials for conveniently and effectively monitoring the tissue-specific distribution of expressed transgenes in cells, tissues, animals or human patients without the need for disruptive sampling methods including surgery.
As used herein, xe2x80x9ccell-associated proteasexe2x80x9d refers to any protease within the cell, such as a protease located in the cytoplasm, or within, or associated with an organelle. As used herein, xe2x80x9ccell-associated proteasexe2x80x9d also refers to any protease associated with the cell, including, but not limited to a protease located on the cell surface or in the extracellular space near the cell surface, such that the protease cleaves a peptide with the appropriate sequence near the cell surface.
As used herein, xe2x80x9cmammalxe2x80x9d refers to any warm blooded organism of the class Mammalia, including, but not limited to rodents, feline, canine, or ungulates. In preferred embodiments of the invention, a xe2x80x9cmammalxe2x80x9d is a human.
As used herein, xe2x80x9ctransgenexe2x80x9d refers to any nucleic acid sequence introduced into a cell and which encodes a polypeptide of interest. As used herein a xe2x80x9ctransgenexe2x80x9d can be a gene which is endogenous to the mammal of the present invention, and which may or may not be endogenously expressed by the cells of the invention into which it is introduced. According to the present invention, a xe2x80x9ctransgenexe2x80x9d can be applied to remedy a disease condition in the process known as gene therapy.
As used herein, xe2x80x9cauto-cleaving sequencexe2x80x9d refers to a short polypeptide sequence of between 10 and 20 amino acids, but preferably between 12 and 18 amino acids, but more preferably between 15 and 17 amino acids, in which cleavage of the propeptide at the C-terminus occurs cotranslationally in the absence of a cell associated protease. Moreover, cleavage can occur in the presence of heterologus sequence information at the 5xe2x80x2 and/or 3xe2x80x2 ends of the xe2x80x9cauto-cleaving sequencexe2x80x9d. An example of an xe2x80x9cauto-cleaving sequencexe2x80x9d useful in the present invention is the that of the foot and mouth disease virus (FMDV) 2A propeptide, in which cleavage occurs at the C-terminus of the peptide at the final glycine-proline amino acid pair. Cleavage of FMDV 2A propeptide is independent of the presence of other FMDV sequences and can generate cleavage in the presence of heterologous sequences. Insertion of this sequence between two protein coding regions results in the formation of a self-cleaving chimera which cleaves itself into a C-terminal fragment which carries the C-terminal proline of the 2A protease on its N-terminal end, and an N-terminal fragment that carries the rest of the 2A protease peptide on its C-terminus (P. deFelipe et al., Gene Therapy 6: 198-208 (1999)). Thus, instead of using a cleavage signal recognizable by a cell-associated protease, the self-cleaving FMDV 2A protease sequence can be employed to link the NIS to the polypeptide encoded by the transgene, resulting in spontaneous release of the NIS from the polypeptide encoded by the transgene.
As used herein, a xe2x80x9cfusogenic polypeptidexe2x80x9d refers to a membrane glycoprotein, including, but not limited to Type I and Type II membrane glycoproteins, which kill cells on which they are expressed by fusing the cells into a partial or complete multinucleated syncytia, which die by sequestration of cell nuclei and subsequent nuclear fusion. Examples of xe2x80x9cfusogenic polypeptidesxe2x80x9d include, but are not limited to gibbon ape leukemia virus and measles virus H glycoprotein.
As used herein, xe2x80x9cdetectingxe2x80x9d refers to the use any in vivo, ex vivo, or in vitro imaging technique capable of measuring a radio-labeled moiety, including, but not limited to standard single positron emission computed tomography (SPECT) or positron emission tomography (PET) imaging systems, used to measure the amount of labeled iodine in a mammal. Labeled iodine of the present invention is xe2x80x9cdetectedxe2x80x9d if the levels of labeled iodine measured following administration of one or more of the nucleic acid constructs described above, or the host cells transfected with one or more of the nucleic acid constructs described above are at all higher than the levels measured prior to administration. Labeled iodine of the present invention is also xe2x80x9cdetectedxe2x80x9d if it is localized to one or more organs, tissues, or cells following the administration of one or more of the nucleic acid constructs described above, or the host cells transfected with one or more of the nucleic acid constructs described above, that it was not localized to prior to the administration of the constructs or cells. According to the invention, labeled iodine is xe2x80x9cdetectedxe2x80x9d if the quantitative or semi-quantitative measurements of labeled iodine yield levels which are between 0.001-90% of the administered labeled iodine dose, preferably between 0.01-70%, preferably between 0.1-50%, more preferably between 1.0-20%, more preferably between 5-10% of the administered labeled iodine dose. In a preferred embodiment, the concentration of labeled iodine in organs, tissues, or cells can be determined by comparing the quantity of labeled iodine measured by methods of the invention, including, but not limited to SPECT or PET, to a standard sample of known labeled iodine concentration.
As used herein, xe2x80x9ctransportedxe2x80x9d refers to the movement of labeled iodine from the outside of one or more cells to the inside of one or more cells as a result of the expression of an NIS by the cell or cells which xe2x80x9ctransportedxe2x80x9d the labeled iodine. Labeled iodine is considered to be xe2x80x9ctransportedxe2x80x9d if the measured levels of iodine in organs, tissues or cells of the invention are between 0.001-90% of the administered labeled iodine dose, preferably between 0.01-70%, preferably between 0.1-50%, more preferably between 1.0-20%, more preferably between 5-10% of the administered labeled iodine dose.
As used herein, the biological activity of an NIS polypeptide, refferd to herein as xe2x80x9cNIS activityxe2x80x9d or xe2x80x9cNIS functionxe2x80x9d is the transport or sequestration of iodine across the cell membrane, i.e., from outside a cell to inside a cell. NIS is an intrinsic membrane glycoprotein with 13 putative transmembrane domains which is responsible for the ability of cells of the thyroid gland to transport and sequester iodide. AN NIS polypeptide useful in the invention with xe2x80x9cNIS activityxe2x80x9d or xe2x80x9cNIS functionxe2x80x9d thus is a membrane glycoprotein with a transmembrane domain and is capable of transporting iodine if the polypeptide is present in a thyroid cell, and can also transport iodine in a non-thyroid cell type described herein.
As used herein, xe2x80x9ca sequence encoding an NISxe2x80x9d, or an xe2x80x9cNIS genexe2x80x9d refers to a nucleotide sequence encoding a polypeptide having the activity of a sodium iodide symporter (NIS). Examples of NIS nucleotide sequences and amino acid sequences include, but are not limited to, SEQ ID Nos 1 and 3 and SEQ ID Nos 2 and 4 respectively, as shown in FIGS. 1-4. NIS nucleotide and/or amino acid sequences also include, but are not limited to homologs or analogs of the nucleotide and/or amino acid sequences of FIGS. 1-4, wherein xe2x80x9chomologsxe2x80x9d are natural variants of NIS which retain NIS activity, and xe2x80x9canalogsxe2x80x9d are engineered variants of NIS which retain NIS activity.
An advantage of the present invention is that the transgene location can be monitored with out adversely affecting the mammal or the cell. That is, NIS is a self-protein, and as such does not stimulate a host immune reaction. Furthermore, the NIS functions solely to sequester iodine into a cell, which does not adversely affect normal cellular function, or overall cell biology.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.